HRP20040430A2 - Process for reacting alkaloids and use of the reaction products in the preparation of medicaments - Google Patents

Description

Field of invention

This invention relates to new methods of reacting alkaloids with phosphorus derivatives and the use of the resulting reaction products.

Background of the Invention and Prior Art

Alkaloids are known for their biological activity in foreign organisms. For example, the milk of Chelidonium maius L. (rosopas) has long been known in folk medicine for the treatment of warts. Chelidonium maius L. contains more than 30 alkaloids, of which helidonine makes up to 80% of the total amount.

If a cytostatic or carcinogenic derivative of phosphorus is added to an alkaloid, synthesis products are obtained that are less toxic than the starting materials but have cytotoxic activity against certain cancerous cell lines.

DE 2 028 330 and US 3 865 830 describe the preparation of triphosphoramide-isoquinoline adducts by reacting selected alkaloids of Chelidonium maius L. with tris(1-aziridinyl)phosphine sulfide in an organic solvent.

AT 354 644 and AT 377 988 describe processes for the preparation of phosphorus derivatives by reacting carcinostatic phosphorus compounds which are available in a water-soluble form by conversion to their salt. The disadvantage of the described procedures is that the conversion of the reaction products into a salt that is soluble in water is not complete and that most of the reaction products are insoluble in water.

US 5 981 512 describes the use of substances described in AT 377 988 and AT 354 644 for the treatment of damage resulting from radiation.

The compounds described in the mentioned invention applications have different cytostatic and carcinostatic activity. The mixture of alkaloids, especially the total alkaloids of Chelidonium maius L. proved to be especially promising therapeutically, the pharmacological activity of which was shown in several studies for the treatment of cancer. The composition of the preparation was found to be important for the clinical effect. In accordance with the phytochemical and phytotherapeutic profession, it is assumed that not only individual ingredients, but the entire mixture of reacted alkaloids are pharmacologically active.

The rest of the unreacted reagent after the reaction was removed from the synthetic mixture. Since tris(1-aziridinyl)phosphine sulfide is soluble in organic solvents, such as benzene, ether or chloroform, in the procedures, in accordance with the previous state of the art, it is proposed to remove unreacted tris(1-aziridinyl)phosphine sulfide from the synthetic mixture by washing of reaction products with ether.

Short description of the pictures

Figure 1 describes the HPLC plot with the characteristic total composition of alkaloids from the roots of Chelidonium maius L.

Figure 2 describes the HPLC fingerprint of the preparation, according to Example 1.

Figure 3 shows selected phosphorus derivatives that are suitable reagents.

Detailed description of the invention

The aim of this invention is to provide a new process for the preparation of the reaction products of alkaloids with phosphorus derivatives, which promotes the conversion of alkaloids into a water-soluble form. This means that, in addition to the selected alkaloids, the total alkaloids of the Chelidonium maius L. plant can also be converted, and that a pharmaceutical preparation with low toxicity and as wide an action spectrum as possible can be obtained.

This object is achieved by the features of Claim 1. Additional embodiments and developments of the present invention are set forth in the subclaims.

The process according to the present invention consists of reacting an alkaloid or a mixture of alkaloids in an organic solvent with phosphorus derivatives and then washing the reaction product with water. The phosphorus derivative preferably contains at least one aziridine group. The washed reaction product can then be converted into a water-soluble form. The phosphorus derivative is soluble in water, especially if it is toxic.

As a result of washing with water, it is possible to significantly simplify the preparation process, since complex safety measures due to the risk of explosion do not need to be taken. As a consequence, the process can also be carried out without problems of industrial importance. In addition, the composition of the reaction product, which is decisive for the pharmacological activity, is changed by the washing process because, with the liquid-liquid separation, the ingredients that are soluble in water dissolve from the reaction product and are thus removed. Surprisingly, water has been found to have an additional catalytic effect on the reaction products, thus influencing the structure and composition of the synthesis product, with 10 to 15 times more of the reaction product being able to be converted to a water-soluble form than by washing with an organic solvent. .

By washing with water, the reaction product is converted into a state that significantly facilitates the conversion into a form that is soluble in water. This measure is therefore appropriate for Chelidonium maius L. alkaloids as well as for alkaloids from other sources. This process can be used, for example, to react alkaloids with carcinostatic phosphorus compounds, as mentioned in Claim 1 AT 377 988, phosphorus derivatives shown in Figure 3, especially those having an aziridine group.

A suitable organic solvent is any agent in which soluble alkaloids are intended to react. Alkaloids can, for example, be dissolved in dichloromethane or chloroform.

The reaction of alkaloids takes place at an elevated temperature, preferably at the boiling temperature of the solvent.

The reaction product is preferably converted into a water-soluble form after washing with water. This can be carried out according to the procedures described in AT 377 988 and AT 354 644 by conversion to salts which are soluble in water, in particular to hydrochlorides, for example by passing through gaseous HCl or by adding a solution of HCl to the organic solution of the washed reaction product, after which precipitates hydrogen chloride. The water-soluble salt of the reaction product is suitable for use in injection solutions.

In one embodiment of the invention, the reaction is carried out with tris(1-aziridinyl)phosphine sulfide (CAS No. 52-24-4), which is known in the pharmacopoeia as thiotepa. Other synonyms are ledertepa, Onco thiotepa, TESPA, tespamine, thiophosphamide, thio-TEPA, thiotriethylenephosphoramide, typhosil, triaziridinylphosphine sulfide, N,N',N''-tri-1,2-ethanediylphosphorothionine triamide; N,N',N''-tri-1,2-ethanediylthiophosphoramide, tri-(ethyleneimino)thiophosphoramide; N,N',N''-triethylenethiophosphoramide, triethylenethiophosphorotriamide, m-triethylenethiophosphoramide, m-tris(aziridin-1-yl)phosphine sulfide, triethylenethiophosphoramide, tris(1-aziridinyl)phosphine sulfur, tris(ethyleneimino)thiophosphate, TSPA and WR 45312.

In the following embodiment of the invention, alkaloid extracts, optionally the total alkaloids of Chelidonium maius L., are reacted with tris(1-aziridinyl)-phosphine sulfide in an organic solvent, and the resulting reaction product, optionally present as a complex, is then washed at least once with water . As tris(1-aziridinyl)-phosphine sulfide decomposes in water, the unconverted residual tris(1-aziridinyl)-phosphine sulfide, present in excess after the reaction, can be removed from the organic phase by this measure. Preferably, the organic solution containing the reaction product is washed several times and saturated with water. It is particularly desirable that the washing is repeated until the excess of the highly toxic tris(1-aziridinyl)-phosphine sulfide is completely removed from the reaction product.

In addition, toxic alkaloids that can contribute to various reactions in medical applications and can even cause liver cirrhosis are removed from the synthetic mixture by the aqueous phase or their concentrations can be reduced. Using the Ames test, it was proven that the reaction product of this embodiment, prepared in accordance with the invention, is not mutagenic.

This reaction product is a complex mixture of alkaloids with a high molecular weight of the reaction products of tris(1-aziridinyl)-phosphine sulfide with alkaloids and the breakdown products of tris(1-aziridinyl)-phosphine sulfide. As a result of the synthesis process, the solubility of alkaloids changes. The concentration of tertiary alkaloids increases while the concentration of quaternary alkaloids decreases. The reaction product consists of a complex of about 60 to 70% Chelidonium alkaloids with about 30 to 40% tris(1-aziridinyl)-phosphine sulfide reaction products.

Tertiary Chelidonium alkaloids represent the main part of the ingredients. For example, the following tertiary alkaloids may be contained in the synthetic mixture: helidonine, protopine, stylopine, allocryptopine, α-homochelidonine, helamidine, helamin, L-sparteine, helidimerine, dihydrosanguinarine, oxysanguarin, oxychelidonine and methoxychelidonine.

Quaternary alkaloids (eg, berberine) were largely removed from the synthetic mixture with water by liquid-liquid partitioning. Under the specified reaction conditions, berberine does not further form compounds with tris(1-aziridinyl)-phosphine sulfide.

The reaction product of this embodiment also exhibits a better medicinal action spectrum than the ether-washed reaction product. It destroys cancer cells by apoptosis but, unlike most known cytostatic agents, without attacking healthy cells. This results in good tolerance of therapy with this preparation and its general suitability for prophylactic use. It is easy to use and there are no significant negative phenomena during therapeutic dosages. The interaction of the ingredients in the synthetic mixture is responsible for the medicinal effect.

The reaction product of total alkaloids Chelidonium maius L. shows biological activity in regulating metabolism and is suitable for the prevention and therapy of metabolic diseases, such as osteoporosis but also rheumatic diseases, allergies, viral infections, epilepsy, multiple sclerosis, scars, skin tumors and postoperative wounds .

The extract of the dried root of Chelidonium maius L. is preferably used as the starting material for the synthesis. The roots have a higher alkaloid content than the leaves or stems.

The usual pharmaceutical excipients, especially for solutions, for example for injection solutions or infusion solutions or for ointments, compresses or suspensions are suitable for medicaments containing the reaction products prepared in accordance with the present invention.

The following examples illustrate the invention:

Example 1

Alkaloid extraction:

25 g of the alkaloid salt mixture was suspended in water and transferred to a separatory funnel. After adding 100 ml of dichloromethane, the separatory funnel is shaken. After that, the organic phase was separated and filtered into a glass bottle.

1N NaOH (pH 8-9) was added to the aqueous phase until cloudy. After adding 100 ml of dichloromethane, the mixture was shaken. After that, the organic phase was separated and combined with the dichloromethane phase from step a. This procedure was repeated, for example 3 times. The organic phases were filtered and combined.

The pH of the aqueous phase was adjusted to pH 10 by adding NaOH. After adding dichloromethane, the mixture was shaken. After that, the organic phase was separated, filtered and mixed with other organic phases. Then the pH of the aqueous phase was adjusted to 13 and the extraction was repeated with dichloromethane.

The combined organic phases were evaporated to give an oily, brown solid.

Reaction with tris(1-aziridinyl)-phosphine sulfide:

The alkaloid residue was dissolved in dichloromethane and tris(1-aziridinyl)-phosphine sulfide was added. The mixture was refluxed at 80°C for 2 hours. After cooling to room temperature, the reaction mixture was purified. After that, the mixture was filtered and the filtrate was washed several times, for example 3 times or more, with 250 ml of water in a separatory funnel.

Reaction with HCl

The washed solution was transferred to a glass beaker, stirred and saturated with HCl gas to precipitate the hydrochloride complex. The precipitated product was filtered off and washed with diethyl ether, dried and then dissolved in water.

From the reaction product according to Example 1, an LD50 value of 485 mg/kg was determined in rats. Research on mice has shown that the product in accordance with this invention changes the hormonal regulation of the mammary gland and stimulates the synthesis of substances that have an activity similar to thymosin in animals whose mammary glands have been removed. This effect is dose dependent. The preparation increases the number of T-lymphocytes in the peripheral blood by up to 50% (4.04 ± 0.43 × 109/l before treatment, 6.24 ± 0.73 × 109/l after treatment), changes the humoral immune response to the penetrating antigen and natural killer activity of spleen cells (198.20 ± 17.69% compared to 71.50 ± 9.10% in the control group) and increases the interferon release potential of white blood cells during animal experiments. The results of animal experiments are confirmed by clinical observations. Therefore, an improvement in immune parameters has been observed in cancer patients.

Doses of about 5 mg of the preparation from Example 1 per 70 kg of body weight can be used for prophylactic and immunological applications. For the treatment of cancer, 5 mg of the preparation per 20 kg of body weight is preferably given.

Example 2:

HPLC fingerprints

The determination was performed by reversed-phase chromatography of the ion pair in a gradient and by means of spectral measurements using a DAD detector at 285 nm. At the same time, chromatograms were prepared using reference alkaloids. In addition, HPLC-MSD analysis was performed, which showed that there were no peaks other than those from alkaloids. The HPLC diagrams in Figures 1 and 2 were obtained on the basis of the following experimental data:

Chromatographic parameters:

Column: LiChrospher 60 RP choice B, 5um, 125 × 24 mm ID

Eluent:

A) 200 ml (acetonitrile) + 800 ml (water) + 1.5 g (hexanesulfonic acid) + 0.3 ml (85% phosphoric acid)

B) 900 ml (acetonitrile) + 100 ml (water) + 1.5 g (hexanesulfonic acid) + 0.3 ml (85% phosphoric acid

Gradient: 5 min isocratic 100% A;

up to 40% B in 24 min

up to 100% B in 1 min

5 min 100% B;

5 min of balance with 100% A

Determination: UV light at 285 nm

Eluent flow rate: 1ml/min, stop after 35 min.

Injected volume: 10 μl

Sample preparation:

Extraction before the reaction (Figure 1): 25 mg of alkaloids were ultrasonically dissolved in 40 ml of methanol, diluted to 50 ml and filtered through a membrane filter.

Reaction product (picture 2): The reaction product was converted into a hydrochloric salt, dissolved in water at a concentration of 1 mg/ml and the pH adjusted to a value between 2.5 and 6.5.

The following examples show different applications of the compound (hereinafter Ukran®) which is produced by the process described in example 1.

Example 3:

Osteoporosis type I and II:

30 patients with type I and II osteoporosis were treated with Ukrain® drug. Doses of 5 mg at a concentration of 1 mg/ml were given intravenously once a week for 10 weeks. The progress of the therapy is monitored by determining the hematological and biochemical parameters and by measuring the concentrations of FSH, LH and prolactin in the serum. Calcium and phosphorus were measured in urine. After three months of treatment, an improvement in the clinical picture and increased physical activity was observed in all patients. No negative effect of the treatment was observed.

Example 4:

Brittle bone disease (Osteogenesis imperfecta):

Type I brittle bone disease was diagnosed in a 14-year-old female patient. X-rays indicated diffuse osteoporosis with the appearance of blue sclera, baby teeth (dentiogenesis imperfecta), chicken breasts, scoliosis and muscle hypotension. Ukrain® was administered intravenously to the patient in doses of 5 mg once a week. Three cycles of therapy were carried out with three-month breaks between cycles. The treatment achieved a significant improvement in muscle function; the sclera and teeth became white again. The scoliosis improved and the intensity of the physical therapy could be significantly increased due to greater load capacity. The patient's body became proportional again. Body weight and body size again became matched to the patient's age. The X-rays indicated restoration of the bone structure, and there were no noticeable signs of osteoporosis on the X-rays.

Example 5:

Spondyloarthritis:

A 71-year-old patient complained of severe pain in the lower back, L1 to L3 area, with reduced mobility. The patient was treated with the drug Ukrain® in doses of 5 mg administered intravenously once a week, as well as with compresses that were applied topically for four weeks. The patient's condition improved and mobility was fully restored.

Example 6:

Skin damage that causes ulceration:

A seven-year-old boy had extensive postoperative skin damage in the area of the left scapula. The scar was too large to be removed by cosmetic surgery. The patient was superficially treated with compresses containing Ukrain®, in daily doses of 5 mg, for 3 weeks. His condition changed significantly and the patient was cured.

Example 7:

Allergic asthma:

Severe allergy with coughing and asthmatic attacks was observed in a 12-year-old patient. Treatment with the drug Ukrain® was carried out in daily doses of 5 mg for 3 weeks. His condition has changed significantly and it is considered that the patient has been cured.

Example 8:

Milk allergy:

A 12-year-old patient suffered from a strong allergy to milk, with the fact that even products containing milk, such as chocolate, caused allergic attacks. The patient was given intramuscularly Ukrain® every other day for 2 weeks. His condition has significantly improved and it is considered that the patient has been cured.

Example 9:

Allergy to cats:

A 9-year-old girl suffered from an allergy to cats that caused severe asthma attacks. The patient was treated with the drug Ukrain® in a dose of 5 mg twice a week. Her condition has improved and she is considered cured.

Example 10:

Shingles:

A 64-year-old patient was diagnosed with herpes zoster infection, which kept coming back. Common blisters were found with damage in the Th3-Th4 area, along with ulceration. Ukrain® was administered intravenously at a dose of 5 mg twice a week for 8 weeks. In the following 4 years, the disease did not return.

Example 11:

Hepatitis C:

A 42-year-old patient with chronic hepatitis C, who was positive for HCV-RNA and with HCV genotype 1b, complained of fatigue accompanied by pain and poor physical endurance. He was given Ukrain® intravenously at a dose of 5 mg twice a week for 5 weeks. After the treatment, the pain disappeared, his general condition improved and his physical endurance increased. The patient is currently HCV-RNA negative.

Example 12:

Allergy:

12-year-old patient A. N. had severe allergic attacks accompanied by cough, including asthmatic episodes. He was treated with the drug Ukrain®, which was given orally in doses of 5 mg per day. After 4 weeks there was a dramatic improvement and no more attacks.

Example 13:

Postoperative wounds (skin damage with ulceration):

7-year-old patient S. D. had extensive skin damage on his left shoulder following surgery. The skin damage was too great for aesthetic reconstruction. The patient was treated locally with Ukraina® compresses for 3 months. The skin damage has significantly decreased in size without suppuration and inflammatory process.

Example 14:

Osteoporosis:

68-year-old patient H.J. had problems with walking and fatigue. After a detailed clinical and laboratory examination, type I osteoporosis was diagnosed. After 10 weeks of treatment with Ukraina®, weekly 5 mg intravenously, the patient felt a subjective improvement confirmed by significant changes in laboratory results regarding bone density and calcium and hormone levels.

Example 15:

Shingles:

37-year-old patient K.R., who overcame chicken pox at the age of 7, had the usual growths on the waist area - Herpes zoster. After treatment with 5 mg of Ukraina® intravenously, twice a week for 4 weeks for the next 6 years, no disease appeared.

Example 16:

Hepatitis B

48-year-old patient G. H. suffered from weakness, fatigue and reduced vitality. After a detailed clinical and laboratory examination, the diagnosis was as follows: chronic hepatitis B, the patient was positive for HBs-Ag and had increased levels of enzymes in the blood. After treatment with Ukrain®, 5 mg intravenously once a week for 3 months, there was a visible subjective improvement and increased vitality. Laboratory tests indicated a significant decrease in the amount of the virus and normalization of the enzyme level in the blood.

Example 17:

Rheumatic diseases, osteoarthritis

71-year-old patient I. N. had severe back pain in the L1-L3 region with reduced mobility. The diagnosis after a detailed clinical and radiological examination was as follows: osteoarthritis. Treatment: Ukrain®, 5 mg intravenously twice a week for 6 weeks. The pain was significantly reduced and the patient achieved increased mobility compared to the reference state.

Example 18:

Seizures of epilepsy

46-year-old patient D.R. had complex focal epileptic seizures with a tendency to worsen symptoms. He was given high doses of phenytoin with adenopathy as a side effect. After treatment with 10 mg of Ukraina® intravenously twice a week for 6 weeks, a significant alleviation of symptoms was noted, and the attacks became rarer and less pronounced. Much lower doses of antiepileptic drugs could be used.

Example 19:

Multiple sclerosis

36-year-old patient M. G. felt inexplicable weakness in his legs and fainting. The intervals between episodes were getting shorter. After a detailed examination, multiple sclerosis was diagnosed. Physical therapy was used without success. After treatment with 5 mg of Ukraina® intravenously twice a week for 4 months, an increase in the interval between attacks was noted, the patient had more strength in his legs and did not feel faint.

Example 20:

Scars:

Patient I.N. had a large hard scar in the abdominal area after abdominal surgery. After treatment with 5 mg of Ukraina® intravenously twice a week for 4 weeks and locally in the form of compresses, a clear positive cosmetic effect was noted.

Example 21:

Flu:

The 24-year-old patient B.N. had annual influenza infections in some cases with pneumonia. Ukrain® was used as a prophylactic measure: 5 mg intravenously twice a week, a total dose of 50 mg in the period before the outbreak of the epidemic. The patient did not get the flu for four years after that.

Example 22:

Diabetes mellitus type II:

62-year-old patient B.L., who had diabetes mellitus type II (non-insulin-dependent diabetes), was treated with a daily dose of 10 mg of glipzide (an antidiabetic drug from the sulfonylurea group). The patient had high blood pressure, overweight despite dieting, exhaustion, fatigue and poor stamina. After treatment with 5 mg of Ukraina® intravenously twice a week for 2 months, the general condition of the patient significantly improved, endurance increased, body weight decreased. The administration of Glipzid was gradually stopped and the patient feels well.

Example 23:

Rehabilitation:

74-year-old patient O. L. had a fracture of the left hip joint. After a major operation that lasted several hours with joint implantation, the patient was bedridden. After treatment with 5 mg of Ukraina® intravenously twice a week for 6 weeks, the general condition of the patient visibly improved, and the rehabilitation time significantly shortened.

Example 24:

Allergy to chocolate:

8-year-old patient V.P. was allergic to chocolate. After treatment with 5 mg of Ukraina® administered orally 3 times a week for 4 weeks, allergic attacks did not occur.

Example 25:

Rehabilitation:

78-year-old patient J.R. had surgery for gallstones and cholecystitis (cholecystectomy). After the operation, Ukrain® was given orally, 5 mg 3 times a week for 2 weeks. A quick and harmless recovery was recorded without any complications. The patient was discharged from the hospital ahead of schedule.

All the applications shown in examples 3 - 25 can be successfully carried out - albeit in some cases with reduced efficiency - with the compounds described in AT 377 988.

Claims (14)

1. Alkaloid reaction procedure, characterized by the fact that it contains steps: preparation of reaction products by the reaction of at least one alkaloid with a phosphorus derivative, which contains at least one aziridine group, in an organic solvent, at an elevated temperature, especially at the boiling point of the solvent, and washing the reaction product obtained in step a); 2. The method according to Claim 1, characterized in that the phosphorus derivative is tris(1-aziridinyl)phosphine sulfide (CAS 52-24-4). 3. The method according to Claim 1, characterized in that the alkaloid is selected from the alkaloids contained in the plant Chelidonium maius L. 4. The method according to Claim 1, characterized in that a mixture of alkaloids reacts. 5. Process according to Claim 4, characterized in that the mixture of all alkaloids of the plant Chelidonium maius L reacts. 6. Process according to Claim 1, characterized in that the solvent is dichloromethane. 7. The method according to Claim 1, characterized in that the reaction products after washing are converted into a salt, especially a water-soluble salt. 8. Process according to Claim 7, characterized in that the salt is chloride. 9. An alkaloid reaction product, which can be obtained by a process according to any one of Claims 1 to 8, characterized in that the reaction product which is present after washing with water: contains an alkaloid derivative obtained from the reaction of a tertiary alkaloid with a phosphorus derivative containing at least one aziridine group; has such properties that it significantly facilitates, preferably by a factor of 10 to 15, the subsequent conversion into a water-soluble salt; it has a better spectrum of medical activity than the reaction product that was washed with ether and preferably destroys cancer cells, but not healthy cells; you has a reduced amount or does not contain toxic alkaloid that is soluble in water or compounds resulting from the decomposition of phosphorus derivatives. 10. Alkaloid reaction product according to Claim 9, characterized in that the mixture containing the alkaloid is the product of the reaction of the alkaloid with a phosphorus derivative, which is preferably tris(1-aziridinyl)phosphine sulfide, and a decomposition product of the said phosphorus derivative. 11. Alkaloid reaction product according to Claims 9 or 10, characterized in that at least one alkaloid is selected from the group consisting of helidonine, protopine, stylopine, allocryptopine, homochelidonine, sanguinarine, helamidine, helamine, L-sparteine and oxychelidonine. 12. Use of an alkaloid reaction product as a medicine, characterized by the fact that it is defined by any of Claims 9 to 11. 13. Use in accordance with Claim 12, characterized by the fact that it is for the production of a preparation for the prophylaxis or treatment of diseases or organ damage selected from the group consisting of cancer, immune diseases or metabolic diseases. 14. Use according to Claim 13, characterized in that the disease is selected from the group consisting of allergies, osteoporosis, skin tumors, viral infections, rheumatic diseases, scars, postoperative wounds, epilepsy and multiple sclerosis.